Quality control reagents and methods

ABSTRACT

The present invention provides reagents for instrumentation quality control and methods of use thereof. In particular, sets of peptides or other molecules are provided for evaluating the performance of instruments with mass spectrometry (MS) and/or liquid chromatography (LC) functionalities.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/275,417, filed Feb. 14, 2019, which divisional of U.S.patent application Ser. No. 14/180,124, filed Feb. 13, 2014, now U.S.Pat. No. 10,247,711, which claims priority to U.S. Provisional PatentApplication Ser. No. 61/764,312 filed Feb. 13, 2013; each of which ishereby incorporated by reference in its entirety.

FIELD

The present invention provides reagents for instrumentation qualitycontrol and methods of use thereof. In particular, sets of peptides orother molecules are provided for evaluating the performance ofinstruments with mass spectrometry (MS) and/or liquid chromatography(LC) functionalities.

BACKGROUND

Mass spectrometry provides a rapid and sensitive technique for thedetermination of the molecular mass of a molecule or mixture ofmolecules. In the analysis of peptides and proteins, mass spectrometrycan provide detailed information regarding, for example, the molecularmass (also referred to as “molecular weight” or “MW”) of the originalmolecule, the molecular masses of peptides generated by proteolyticdigestion of the original molecule, the molecular masses of fragmentsgenerated during the ionization of the original molecule, and evenpeptide sequence information for the original molecule and fragmentsthereof. Mass spectrometers are extremely precise; their performance andcalibration must be carefully monitored as systematic errors may causeerroneous m/z values or changes in sensitivity. Methods and kits for thecalibration of mass spectrometers have been described in various patentsand applications (See, e.g., U.S. Pat. No. 4,847,493; U.S. PatentApplication Publication No. 2002/0033447; U.S. Patent ApplicationPublication No. 2002/0045269; U.S. Patent Application Publication No.2003/0062473) and are also commercially available (See, e.g., PROTEOMASSPeptide and Protein MALDI-MS Calibration Kit (Sigma-Aldrich, St. Louis,Mo., USA); Mass Standards Kit (Applied Biosystems, Foster City, Calif.,USA); MASSPREP reference standards (Waters, Milford, Mass., USA);Protein Calibration Standard I 20,000-70,000 Da (Bruker Daltonics,Billerica, Mass., USA); All-in-1 Protein Standard (Ciphergen, Fremont,Calif., USA)). However, these kits do not directly provide a measure ofinstrument sensitivity or dynamic range in a single run.

SUMMARY

In some embodiments, the present invention provides reagents comprisingtwo or more distinct-mass versions of each of two or moredistinct-structure (e.g., sequence) molecules (e.g., peptide, nucleicacid, peptide nucleic acid, polymer, etc.). Although many embodiments ofthe present invention are described as comprising or for use withpeptide reagents, these embodiments should be viewed more broadly asapplying to quality control and performance evaluation reagentscomprising other molecules and/or polymers. In some embodiments, thepresent invention is not limited to peptide reagents.

In some embodiments, the present invention provides peptide mixturescomprising two or more distinct-mass versions of each of two or moredistinct-sequence peptides. In some embodiments, the present inventionprovides peptide mixtures consisting of, or consisting essentially of,two or more distinct-mass versions of each of two or moredistinct-sequence peptides. In some embodiments, each of thedistinct-sequence peptides is of distinct hydrophobicity. In someembodiments, all of the distinct-mass versions of any of thedistinct-sequence peptides are present at distinct concentrations. Insome embodiments, the distinct-mass versions of any of thedistinct-sequence peptides are present at concentrations ranging from atleast as low as 0.1 nM to at least as high at 100 μM. In someembodiments, the distinct-mass versions of any of the distinct-sequencepeptides are present at concentrations ranging from 1 nM to 10 μM. Insome embodiments, the distinct-mass versions of any of thedistinct-sequence peptides are present in a reagent at zeptomoles topicomoles of total peptide. In some embodiments, the distinct-sequencepeptides are separable by liquid chromatography based on their differenthydrophobicities or other chemical properties (e.g., charge, size, orhydrophilicity). In some embodiments, a peptide mixture comprises 3-20distinct-sequence peptides. In some embodiments, a peptide mixturecomprises 5-10 distinct-sequence peptides. In some embodiments, thedistinct-mass versions of any of the distinct-sequence peptides aredifferentiable by mass spectrometry. In some embodiments, thedistinct-mass versions of any of the distinct-sequence peptides are theresult of different combinations of stable isotope-labeled amino acids.In some embodiments, the distinct-mass versions of any of thedistinct-sequence peptides are the result of different combinations ofstable isotope of constituent atoms. In some embodiments, each of thedistinct-mass versions of any of the distinct-sequence peptidescomprises a different number of uniformly stable isotope-labeled aminoacids. In some embodiments, a peptide mixture comprises 3-20distinct-mass versions of each of the distinct-sequence peptides. Insome embodiments, a peptide mixture comprises 5-10 distinct-massversions of each of the distinct-sequence peptides.

In some embodiments, the present invention provides methods forassessing performance of an instrument with both liquid chromatography(LC) and mass spectrometry (MS) functionalities comprising: (a)introducing a peptide mixture to the instrument, wherein said peptidemixture comprises two or more distinct-mass versions of each of two ormore distinct-sequence peptides; (b) analyzing the peptide mixture by LC(consisting of various mobile phases, including but not limited to C₁₈,SCX, HILIC etc.); (c) analyzing the peptide mixture by MS (consisting ofbut not limited to ESI or MALDI methods of ionization); and (d)assessing the performance of the LC and MS functionalities of theinstrument based on results of steps (b) and (c). In some embodiments,the peptide mixture purified peptides. In some embodiments, the purifiedpeptides of the peptide miXture are the only peptides introduced to theinstrument in step (a). In some embodiments, the peptide mixturecomprises peptides in the presence of one or more impurities. In someembodiments, the peptide mixture is introduced to the instrument in thepresence of one or more other peptides (e.g., background, contaminants,etc.). In some embodiments, the peptide mixture is a peptide mixturethat comprises two or more distinct-mass versions of each of two or moredistinct-sequence peptides. In some embodiments, assessing theperformance of the LC and MS functionalities of the instrument comprisesreporting one or more LC-parameters for each peptide sequence selectedfrom: retention times, peak height, peak width, peak resolution, andpeak symmetry or one or more MS-parameters selected from mass accuracy,mass resolution, sensitivity, dynamic range, linear response, MS/MSspectral quality and sampling and/or isolation efficiency. In someembodiments, assessing the performance of the LC and MS functionalitiesof the instrument comprises reporting one or more MS-parameters for eachdistinctly-massed version of one or more of the peptide sequencesselected from: resolution, mass accuracy, and sensitivity as a “neat”mixture, sensitivity within a complex mixture, instrumental sampling,and dynamic range. In some embodiments, the assessment of LC and/or MSperformance is provided by a software program. In some embodiments, thesoftware reports LC and/or MS parameters from a single analysis, theperformance history of the instrument or a comparison between multipleLC and/or MS instruments or instrument platforms. In some embodiments,the software provides a performance score of the LC and/or MSinstrument. In some embodiments, sensitivity and/or performance areevaluated for a neat sample (e.g., reagent without substantialbackground causing agents). In some embodiments, sensitivity and/orperformance are evaluated for a complex sample (e.g., reagent andbackground causing agents/peptides).

In some embodiments, the present invention provides methods forassessing performance of an instrument with both liquid chromatography(LC) and mass spectrometry (MS) functionalities comprising: (a)introducing a peptide mixture to the instrument, wherein said peptidemixture comprises two or more distinct-mass versions of each of two ormore distinct-sequence peptides, wherein said peptide mixture comprisestwo or more distinct-mass versions of each of two or moredistinct-sequence peptides; (b) analyzing the peptide mixture by LC; (c)analyzing the peptide mixture by MS; and (d) assessing the performanceof the LC and MS functionalities of the instrument based on results ofsteps (b) and (c). In some embodiments, distinct-sequence peptides areof distinct hydrophobicities and separable by LC. In some embodiments,the present invention provides distinct-mass versions that comprisedifferent combinations of heavy isotope labeled amino acids and areresolved by MS. In some embodiments, the assessment of LC and MSperformance is provided by a software program. In some embodiments, thesoftware reports LC and MS parameters from a single analysis, theperformance history of the instrument or a comparison between multipleLC and MS instruments. In some embodiments, the software provides aperformance score of the LC and/or MS instrument.

In some embodiments, the present invention provides reagents and/ormethods for quality control and/or evaluating the performance ofinstruments with mass spectrometry (MS) and/or liquid chromatography(LC) functionalities. In some embodiments, both MS and LCfunctionalities are assessed. In some embodiments, only one of MS and LCfunctionalities are assessed. In some embodiments, an instrument hasboth MS and LC functionalities, but only one is assessed. In someembodiments, an instrument has only one of MS and LC functionalities. Insome embodiments, MS and LC functionalities are provided by a singleunit. In some embodiments, MS and LC functionalities are provided byseparate units.

In some embodiments, the assessment of the performance of LC and/or MSfunctionality is performed by software. In some embodiments, thesoftware generates a performance score. In some embodiments, aperformance score is unique to the tested instrument (or type ofinstrument tested). In some embodiments, a performance score can be usedfor assessment of the tested instrument (or type of instrument tested)at a given time and/or over time. In other embodiments, a performancescore is comparable to performance scores of other similar and/ordifferent instruments (e.g., those with LC and/or MS functionality). Insome embodiments, a performance score can be used for comparison of atested instrument (or type of instrument tested) to other similar and/ordifferent instruments at a given time and/or over time. In someembodiments, software is used to track an instruments historicalperformance. In some embodiments, a report of instrument performanceparameters (e.g., score) is generated.

In some embodiments, the present invention provides methods forassessing performance of an instrument with both liquid chromatography(LC) and mass spectrometry (MS) functionalities comprising: (a)introducing (e.g., injecting) a peptide mixture to the instrument,wherein the peptide mixture comprises two or more distinct-mass versionsof each of two or more distinct-sequence peptides; (b) separating oranalyzing the peptide mixture by LC; (c) analyzing the peptide mixtureby MS; and (d) assessing the performance of the LC and MSfunctionalities of the instrument based on results of steps (b) and (c)using software. In some embodiments, the peptide mixture is a peptidemixture that comprises two or more distinct-mass versions of each of twoor more distinct-sequence peptides. In some embodiments, eachdistinct-mass version of a distinct-sequence peptide is present at adifferent concentration. In some embodiments, assessing the performanceof the LC and MS functionalities of the instrument comprises reportingone or more LC-parameters for each peptide sequence selected from:retention times, peak height, peak width, peak resolution, and peaksymmetry. In some embodiments, assessing the performance of the LC andMS functionalities of the instrument comprises reporting one or moreMS-parameters for each distinctly-massed versions of one or more of thepeptide sequences selected from: resolution, mass accuracy, sensitivityof “neat” samples, sensitivity within complex samples, dynamic range,mass resolution, isolation efficiency, MS/MS spectral quality and linearresponse in a single analytical experiment. In some embodiments,performance is assessed by software which generates a performance score.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows an illustration of a chromatogram (top panel) of a reagentcomprising six distinct-sequence peptides, and a mass spectrum (bottompanel) of one of those distinct-sequence peptides which is made up of 5distinct-mass versions. The distinct-sequence peptides are designed tobe spaced (relatively) evenly across a standard chromatographic gradientwhen analyzed, for example, on a C₁₈ reversed phase column using anacetonitrile/0.1% organic acid (like Formic acid or trifluoroaceticacid) gradient. From a chromatographic standpoint it would appear thatthe product only contains six peptides. However, mass spectra revealthat each peak corresponding to a peptide sequence corresponds to fiveisomeric peptides which, although identical in sequence, are clearlydistinguished by a mass spacing of at least 4 Daltons.

FIG. 2 shows a table of five distinct-mass versions of a peptidesequence (SEQ ID NO: 5) and their respective masses (right). Anexemplary mass spectrum for these peptides is provided (left). In someembodiments, mass spacing of 7-10 Daltons is provided, allowing forspacial resolution in both high and low-resolution MS instruments. Inorder to achieve this mass differentiation (i.e. distinct-mass variants)stable, heavy isotopically enriched amino acids are incorporated.

FIG. 3 shows an illustration of an embodiment of the present inventionin which distinct-mass versions of a peptide sequence are provided atdifferent concentrations. Because their quantities are precisely known,individual (distinct mass) peptides can be mixed in ratios such that aplot of relative intensity versus the molar amount on column is linearwhen plotted as the log (fold-difference) of the corrected intensity. Inthe example provided, the lightest peptide (i.e. the peptide with onlyone heavy labeled amino acid) is the most abundant. Each successivelyheavy peptide is 10× lower in concentration, thus giving the desiredlinear relationship.

FIG. 4 shows an illustration of an embodiment of the inventioncomprising five distinct-mass versions of each of six distinct-sequencepeptides. The top panel depicts a chromatogram of the reagent, in whichdistinct peaks are visible for each of the distinct sequences. The lowerpanel depicts mass spectra for each sequence, in which each distinctmass version is separately identifiable. Thus, the whole mixture is a 30peptide mixture, in this embodiment.

FIGS. 5A-B show graphs depicting the effect of loading time on thebinding of the peptides to the LC column.

FIG. 6 shows LC analysis of 5 isotopologue mixes of peptides,demonstrating that each isomeric peptide, regardless of mass, co-elutesfrom the column.

FIG. 7 shows LC analysis of peptide mix using different chromatographicgradients (Buffer A=0.1% formic acid in water and Buffer B=0.1% formicacid in acetonitrile). Regardless of the gradient, separation and peakshape is retained in all cases. From left to right SEQ ID Nos: 1-6.

FIG. 8 shows a graph used to calculate correction factors used forpeptide quantification. Correction factors are required to normalize allpeak intensities to correct for distribution of naturally occurring,heavy isotopes. The correction factor represents the ratio of themonoisotopic m/z intensity to the total isotopic distribution intensityand is used to derive the correct intensity of the ion when all isotopescannot be fully detected.

FIG. 9 shows a representative mass spectrum of five distinct massversions of a single peptide sequence (SEQ ID NO: 5). These masses(which are the doubly charged peptides) are easily resolved on unitresolution instruments. This is particularly clear in this example,albeit at high-resolution.

FIG. 10 shows the results of MS analysis of a peptide reagent comprisingfive distinct-mass versions of each of six distinct-sequence peptides.

FIG. 11 shows linear analysis of the mass spectrum of a reagentcomprising five distinct mass versions of a single peptide sequence.

FIG. 12 shows linear analysis of the mass spectrum of a reagentcomprising five distinct-mass versions of each of six distinct-sequencepeptides.

FIG. 13 shows linear analysis of the mass spectrum of a reagentcomprising five distinct-mass versions of each of six distinct-sequencepeptides (reversed concentrations from FIG. 12).

FIG. 14 shows MS analysis of five distinct mass versions of a singlepeptide sequence (SEQ ID NO: 5), in which each successively heavierversion is present at 10-fold concentration excess over the immediatelylighter version.

FIG. 15 shows MS analysis of a reagent comprising five distinct-massversions of each of six distinct-sequence peptides, in which eachsuccessively heavier version of each peptide is present at 10-foldconcentration excess over the immediately lighter version.

FIG. 16 shows MS analysis of a reagent comprising five distinct-massversions of each of six distinct-sequence peptides, in which eachsuccessively lighter version of each peptide is present at 10-foldconcentration excess over the immediately lighter version.

FIG. 17 shows chromatographic detection of all peptides (SEQ ID NOs:1-6) when spiked into a complex background of a yeast tryptic digest.

FIG. 18 shows the lowest detectable peptide quantity (LDPQ) analysis ofa reagent comprising five distinct-mass versions of each of sixdistinct-sequence peptides in a yeast tryptic digest background(starting concentration 2 fmol). Note that, with this mixture, all ofthe peptides are detectable down to 200 amol, despite being present in ahighly complex mixture.

FIG. 19 shows limit of quantification (LOQ) analysis of a reagentcomprising five distinct-mass versions of each of six distinct-sequencepeptides in a yeast background (starting concentration of 200 fmol).

FIG. 20 shows limit of quantification (LOQ) analysis of a reagentcomprising five distinct-mass versions of each of six distinct-sequencepeptides in a yeast background, following a second dilution (startingconcentration of 20 fmol).

FIG. 21 shows limit of quantification (LOQ) analysis of a reagentcomprising five distinct-mass versions of each of six distinct-sequencepeptides in a yeast background, following a second dilution (startingconcentration of 2 fmol).

DEFINITIONS

As used herein, unless otherwise specified, the term “peptide” refers toa polymer compound of two or more amino acids joined through the mainchain by peptide amide bonds (—C(O)NH—). When used in conjunction with,or in comparison to, the term “polypeptide,” the term “peptide”typically refers to short amino acid polymers (e.g., chains having fewerthan 25 amino acids), whereas the term “polypeptide” refers to longeramino acid polymers (e.g., chains having more than 25 amino acids).

As used herein, the term “distinct-sequence peptides” refers to a groupof peptides (e.g., two or more) that can be differentiated from oneanother by the identity and/or order of their amino acids. For example,‘three distinct-sequence peptides’ are three amino acid chains that,although possibly sharing other characteristics, each comprise at leastone amino acid difference, and possibly many amino acid differences,from each other (e.g., LLSLGALEFK (SEQ ID NO: 7), LSSLGALEFK (SEQ ID NO:8), and AAPGEDSRKY (SEQ ID NO: 9)).

As used herein, the term “distinct-mass peptides” refers to a group ofpeptides (e.g., two or more) that can be differentiated from one anotherby mass, even if they cannot be differentiated by sequence.“Distinct-mass versions of a peptide” refers to a group of peptides thatcan be differentiated from one another by mass, even though they cannotbe differentiated by amino acid sequence (e.g., peptides have the sameamino acid sequence). For example, ‘three distinct-mass versions of apeptide’ are three amino acid chains that, although having the sameamino acid sequence, are tagged or labeled (e.g., stable heavy isotopelabeled) to have different masses (e.g., LLSLGALEFK (SEQ ID NO: 7),L*LSLGALEFK (SEQ ID NO: 7), and L*L*SLGALEFK (SEQ ID NO: 7); wherein *indicates uniform isotopic labeling of the preceding amino acid).

As used herein, the term “isomeric peptides” refers to a group ofpeptides (e.g., two or more) that have the same amino acid sequence. Thepeptides do not differ in terms of tags or chemical modifications, buttypically contain varying degrees of stable, heavy isotope labeling(e.g., ¹³C, ¹⁵N, ¹⁸O, ²H, etc.). A pair of isomeric peptides may differin the number of uniformly ¹³C/¹⁵N-labeled amino acids they contain. Forexample, L*LSLGALEFK (SEQ ID NO: 7) and L*L*SLGA*LEFK (SEQ ID NO: 7) (*indicates uniform ¹³C/¹⁵N labeling of the preceding amino acid) areisomeric peptide (they are also ‘distinct-mass peptides’ and‘distinct-mass versions of a peptide with the sequence LLSLGALEFK (SEQID NO: 7)).

As used herein, the term “heavy,” refers to an isotope of an elementthat has a higher molecular mass than the isotope that is most prevalentat natural abundance (e.g., ¹³C instead of ²H instead of ¹H, ¹⁵N insteadof ¹⁴N, ¹⁸O instead of ¹⁶O, etc.), or any chemical entities (e.g.,peptides, molecules, etc.) comprising one or more of such isotopes.

As used herein, the term “uniformly heavy labeled” refers to a molecularentity (e.g., peptide, amino acid, etc.) in which substantially all ofone or more elements within the molecular entity are present as a heavyisotope instead of the isotope that is most prevalent at naturalabundance. For example, uniformly ¹³C/¹⁵N-labelled amino acid is one inwhich substantially all the carbons and nitrogens(e.g., >95%, >98%, >99%, >99.9%) are present as ¹³C instead of ¹²C and¹⁵N instead of ¹⁴N. In some embodiments, a “heavy labeled peptide” maycomprise one or more uniformly heavy labeled amino acids.

DETAILED DESCRIPTION

The present invention provides reagents for instrumentation qualitycontrol and methods of use thereof. In particular, sets of peptides orother molecules are provided for evaluating the performance ofinstruments with mass spectrometry (MS) and/or liquid chromatography(LC) functionalities.

In particular embodiments, provided herein are reagents for calibration,performance evaluation, performance monitoring, system suitability,quality control (QC), etc. of analytical instruments (e.g., HPLC (withUV or other modes of detection), MS, LC-MS, etc.). In some embodiments,a QC reagent comprises a set of peptides that can be predictablyseparated and/or analyzed by LC, MS, or both. In some embodiments,peptides of reagents provided herein exhibit a broad range ofhydrophobicities or other chemical characteristics such that they can beused to probe the entire separation range of an LC instrument. In someembodiments, peptide reagents provided herein exhibit a broad range ofmasses such that they can be used to probe the mass/charge (m/z) ratiorange of an MS instrument. In some embodiments, by reviewing theanalysis of a reagent of the present invention (e.g., manually orautomated by software), the performance of the analyzing instrument isevaluated. By comparing present and past performance evaluations (e.g.,relative to the same peptide reagent), changes in instrument performancecan be identified, monitored, and/or recorded over periods of timeranging from days to months.

In certain embodiments, the present invention provides reagentscomprising two or more distinct-sequence peptides (e.g., 2 sequences, 3sequences, 4 sequences, 5 sequences, 6 sequences, 7 sequences, 8sequences, 8 sequences, 10 sequences . . . 15 sequences . . . 20sequences . . . 25 sequences . . . 30 sequences . . . 50 sequences, ormore). In certain embodiments, reagents comprise three or moredistinct-sequence peptides, four or more distinct-sequence peptides,five or more distinct-sequence peptides, six or more distinct-sequencepeptides, seven or more distinct-sequence peptides, eight or moredistinct-sequence peptides, nine or more distinct-sequence peptides, tenor more distinct-sequence peptides, twelve or more distinct-sequencepeptides, fifteen or more distinct-sequence peptides, twenty or moredistinct-sequence peptides, etc.

In some embodiments, each peptide sequence of a group ofdistinct-sequence peptides is of a detectably different hydrophobicity.In certain embodiments, each peptide sequence of a group ofdistinct-sequence peptides has a unique hydrophobicity (e.g., eachpeptide is separable from all of the others by LC). In otherembodiments, one or more peptide sequence of a group ofdistinct-sequence peptides has a unique hydrophobicity (e.g., one ormore, but not all, peptides are separable from all of the others by LC).In some embodiments, the differences in hydrophobicity can be detectedby liquid chromatography (e.g., high performance liquid chromatography(HPLC)). In certain embodiments, each distinct-sequence peptide resultsin a chromatography peak that is distinguishable from the peaks of theother distinct-sequence peptide. In some embodiments, thedistinct-sequence peptides span a hydrophobicity range such that theycan all be distinguished (e.g., separated) on a single run through a LCcolumn.

In some embodiments, distinct-mass versions of a peptide are provided.In some embodiments, two or more distinct-mass, same-sequence peptidesare provided. In particular embodiments, distinct-mass versions of thesame peptide sequence comprise different isotopic labeling. For example,distinct-mass versions of the same peptide sequence may comprisedifferent degrees of heavy isotope labeling. In certain embodiments,all, or a portion of, the amino acids in a peptide comprise amountsabove natural abundance levels of heavy isotopes (e.g., ²H, ¹³C, ¹⁵N,¹⁸O, etc.). In some embodiments, same sequence peptides have differentmasses based on the degree of heavy isotope labeling of all or a portionof their amino acids (e.g., all amino acids in a peptide at naturalabundance, all amino acids in a peptide 25% ¹³C/¹⁵N-labelled, all aminoacids in a peptide 50% ¹³C/¹⁵N-labelled, all amino acids in a peptide75% ¹³C/¹⁵N-labelled, all amino acids in a peptide >99%¹³C/¹⁵N-labelled, etc.). In other embodiments, same-sequence peptideshave different masses based on the number of fully (e.g., uniformly¹³C/¹⁵N labeled) heavy isotope labeled amino acids in the respectivepeptides (e.g., 0 amino acids uniformly labeled, 1 amino acid uniformlylabeled, 2 amino acids uniformly labeled, 3 amino acids uniformlylabeled, 4 amino acids uniformly labeled, 5 amino acids uniformlylabeled, 6 amino acids uniformly labeled, or more). In some embodiments,differences in mass of the same sequence peptides result from acombination of the number of isotopically-labeled amino acids and thedegree to which they are labeled (3 amino acids are 50%¹³C/¹⁵N-labelled, all amino acids have 99% non-exchangeable ²H labeled,6 amino acids 75% ¹⁸O labeled, etc.). Mass-tags and other labels ormodifications may also be employed to alter the mass of peptides withoutchanging the amino acid sequence.

In some embodiments, two or more distinct-mass versions of a singlepeptide sequence are provided. In some embodiments, two or more (e.g.,2, 3, 4, 5, 6, 7, 8, 9, 10, or more) distinct-mass versions of each of aplurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) ofdistinct-sequence peptides are provided.

In various embodiments, the distinct-mass versions of a peptide (e.g., adistinct-sequence peptide) are not distinguishable by liquidchromatography (e.g., all distinct-mass versions of a peptide elute froma chromatography column (e.g., HPLC column) in a single peak). Invarious embodiments, all distinct-mass versions of a peptide sequenceexhibit substantially identical hydrophobicity. In some embodimentsisomeric peptides have identical or substantially identicalhydrophobicities and elute from a chromatography column (e.g., HPLCcolumn) in a single peak.

In some embodiments, a set (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)of distinct-mass isomeric peptides are provided. In some embodiments,each isomer has the same or substantially the same hydrophobicity andco-elutes from liquid chromatography (e.g., HPLC). In some embodiments,each isomer contains a distinct pattern (e.g., different number) ofstable isotope labeling (e.g., different combination of uniform ¹³C/¹⁵Nlabeled amino acids), and can therefore be distinguished by massspectrometry. In some embodiments, a distinct-mass isomer in a set isprovided at any suitable concentration (e.g., 10 pM . . . 100 pM . . . 1nM . . . 10 nM . . . 100 nM . . . 1 μM . . . 10 μM . . . 100 μM).Distinct-mass isomers may be of unique concentrations or may be at thesame concentration as one or more other distinct-mass peptides of thesame set. In certain embodiments, each distinct-mass isomer in a set isprovided at the same concentration (e.g., 10 pM . . . 100 pM . . . 1 nM. . . 10 nM . . . 100 nM . . . 1 μM . . . 10 μM . . . 100 μM). In otherembodiments, each distinct-mass isomer in a set is provided at adifferent concentration (e.g., a set of five distinctly-massed isomersprovided at 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM, respectively).

In some embodiments, the present invention provides two or more (e.g.,2, 3, 4, 5, 6, 7, 8, 9, 10, or more) distinct-sequence sets of two ormore (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) distinct-mass isomericpeptides. Table 1 depicts generic peptides comprising sixdistinct-sequence sets (sequences A-F) of four distinct-mass versionseach (0, 1, 2, or 3 labeled amino acids).

TABLE 1 Generic distinct-sequence sets of distinct-mass isomericpeptides. Amino Acid Number of uniformly Sequence labeled amino acidsSequence A 0 Sequence A 1 Sequence A 2 Sequence A 3 Sequence B 0Sequence B 1 Sequence B 2 Sequence B 3 Sequence C 0 Sequence C 1Sequence C 2 Sequence C 3 Sequence D 0 Sequence D 1 Sequence D 2Sequence D 3 Sequence E 0 Sequence E 1 Sequence E 2 Sequence E 3Sequence F 0 Sequence F 1 Sequence F 2 Sequence F 3

While Table 1 depicts 0-3 labeled amino acids per sequence, sets withgreater number of labeled amino acids are contemplated (e.g., 0-5, 0-10,etc.). In some embodiments, the distinct-sequence peptides of a reagentmay each be present as the same number of distinct-mass versions (asdepicted in Table 1). In other embodiments, two or moredistinct-sequence peptides of a reagent comprise different numbers ofdistinct-mass versions.

Table 2 depicts an embodiment of the invention which comprises sixdistinct sequence sets (VTSGSTSSR (SEQ ID NO: 1), LASVSVSR (SEQ ID NO:2), YVYVADVAAK (SEQ ID NO: 3), VVGGLVALR (SEQ ID NO: 4), LLSLGAGEFK (SEQID NO: 5), LGFTDLFSK (SEQ ID NO: 6)) of six distinct mass isomericpeptides each.

TABLE 2 Exemplary distinct-sequence sets of distinct-mass isomericpeptides. Set distinct-sequence peptides distinct-mass isomers SEQ IDNO: 1 VTSGSTSSR V*T*S*GST*ST*SR* 1 1 V*T*SGSTST*SR* 1 1 V*T*SGSTSTSR* 11 V*TSGSTSTSR* 1 1 VTSGSTSTSR* 1 1 VTSGSTSTSR 1 2 LASVSVSRL*A*SV*SV*S*R* 2 2 L*ASV*SV*SR* 2 2 LASV*SV*SR* 2 2 LASVSV*SR* 2 2LASVSVSR* 2 2 LASVSVSR 2 3 YVYVADVAAK YV*YV*ADV*A*A*K* 3 3YV*YV*ADV*AAK* 3 3 YVYV*ADV*AAK* 3 3 YVYVADV*AAK* 3 3 YVYVADVAAK* 3 3YVYVADVAAK 3 4 VVGGLVALR V*V*GGL*V*ALR* 4 4 V*V*GGLV*ALR* 4 4V*V*GGLVALR* 4 4 V*VGGLVALR* 4 4 VVGGLVALR* 4 4 VVGGLVALR 4 5 LLSLGAGEFKL*L*SL*GAGEF*K* 5 5 L*L*SL*GAGEFK* 5 5 L*L*SLGAGEFK* 5 5 L*LSLGAGEFK* 55 LLSLGAGEFK* 5 5 LLSLGAGEFK 5 6 LGFTDLFSK L*GF*TDL*F*SK* 6 6L*GFTDL*F*SK* 6 6 L*GFTDL*FSK* 6 6 L*GFTDLFSK* 6 6 LGFTDLFSK* 6 6LGFTDLFSK 6 *indicates uniform ¹³C/¹⁵N labeling of the preceding aminoacid)

The distinct-sequence sets exhibit varying degrees of hydrophobicity,and therefore each set elutes independently when analyzed by liquidchromatography (e.g., HPLC). Each distinct-mass isomer of adistinct-sequence set has the same hydrophobicity, and thereforeco-elute when analyzed by liquid chromatography (e.g., HPLC). Any othersuitable combinations of distinct-sequence sets of distinct-mass isomersare within the scope of the present invention. In some embodiments, theinvention is not limited by the length, sequence, number, or labeling ofthe peptides.

In some embodiments, peptides within a set of distinct sequence peptidesare selected based on criteria including, but not limited to: peakintensity, peak width, peak compactness (narrowness), LC retention time(e.g., not overlapping with another peptide in the reagent), absence ofexcluded amino acids (e.g., P, M, W, C, N, Q, and/or N-terminal E) whichare deemed to contribute to product instability, feasibility ofincorporating sufficient number (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) of stable, isotope labeled amino acids (e.g., commerciallyavailable stable isotope labeled amino acids), stability, ease ofachieving high degree(e.g., >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99%) ofpurity, etc. In some embodiments, peptides comprise one or moreuniformly isotopically ¹³C/¹⁵N labeled amino acids, for example, thoseselected from Table 3.

TABLE 3 Exemplary universally ¹³C/15N labeled amino acids Molecularformula Molecular formula of ¹³C/15N of ¹³C/15N Three Singleuniversally- universally- Mass shift Letter Letter labeled free labeledamino relative to Code Code amino acid acid residues unlabeled AlaA{circumflex over ( )} (13C)3H7(15N)O2 (13C)3H5(15N)O (+4) ArgR{circumflex over ( )} (13C)6H14(15N)4O2 (13C)6H12(15N)4O (+10)  AsnN{circumflex over ( )} (13C)4H8(15N)2O3 (13C)4H6(15N)2O2 (+6) AspD{circumflex over ( )} (13C)4H7(15N)O4 (13C)4H5(15N)O3 (+5) CysC{circumflex over ( )} (13C)3H7(15N)O2S (13C)3H5(15N)OS (+4) GlnQ{circumflex over ( )} (13C)5H10(15N)2O3 (13C)5H8(15N)2O2 (+7) GluE{circumflex over ( )} (13C)5H9(15N)O4 (13C)5H7(15N)O3 (+6) GlyG{circumflex over ( )} (13C)2H5(15N)O2 (13C)2H3(15N)O (+3) IleI{circumflex over ( )} (13C)6H13(15N)O2 (13C)6H11(15N)O (+7) LeuL{circumflex over ( )} (13C)6H13(15N)O2 (13C)6H11(15N)O (+7) LysK{circumflex over ( )} (13C)6H14(15N)2O2 (13C)6H12(15N)2O (+8) MetM{circumflex over ( )} (13C)5H11(15N)O2S (13C)5H9(15N)OS (+6) PheF{circumflex over ( )} (13C)9H11(15N)O2 (13C)9H9(15N)O (+10)  ProP{circumflex over ( )} (13C)5H9(15N)O2 (13C)5H7(15N)O (+6) SerS{circumflex over ( )} (13C)3H7(15N)O3 (13C)3H5(15N)O2 (+4) ThrT{circumflex over ( )} (13C)4H9(15N)O3 (13C)4H7(15N)O2 (+5) TyrY{circumflex over ( )} (13C)9H11(15N)O3 (13C)9H9(15N)O2 (+10)  ValV{circumflex over ( )} (13C)5H11(15N)O2 (13C)5H9(15N)O (+6)

In some embodiments, provided herein is a reagent (e.g., peptidemixture), and methods of use thereof, for the validation, calibration,performance assessment, and/or performance monitoring of analyticinstruments (e.g., liquid chromatographs with UV and/or MS detection).In some embodiments, a reagent provides a performance meter that is usedto monitor instrument performance. Reagents and methods provide:assessment of initial instrument performance, monitoring of instrumenthistory, comparison of parameters between instruments, validation ofinstrument performance (e.g., before or after use), etc.

In some embodiments, a reagent comprises a plurality (e.g., 5) ofdistinct-sequence sets of several (e.g., 5) distinct-mass isomericpeptides each. The reagent is analyzed by liquid chromatography, whichseparates each of the distinct-sequence sets from each other by theirdifferent hydrophobicities. The reagent is subsequently analyzed by massspectrometry, which characterizes the distinct-mass isomers within eachset by their mass-to-charge ratios. The mass differences (e.g., 3-5Daltons) between the same-sequence isomers are apparent from suchMS-characterization. In some embodiments, the different isomers within aset are provided at a range of different concentrations. Theconcentration differences among the isomers within each set are apparentbased on the MS analysis and/or amino acid analysis. In someembodiments, analysis of a single reagent by both the LC and MScomponents of an instrument provides simultaneous performance analysis.In other embodiments, a reagent is first analyzed by LC and thenanalyzed by MS. In some embodiments, a reagent is analyzed by LC and MSsimultaneously (e.g., two portions of the same reagent are eachsubjected to analysis by one technique).

In some embodiments, all the peptides in a reagent are provided at thesame concentration. In some embodiments, each distinct-mass version of apeptide sequence is the same concentration. In some embodiments, eachdistinct-mass version of a peptide sequence is a differentconcentration. In some embodiments, the distinct-mass versions of apeptide sequence are provided at a range of concentrations. Inparticular embodiments, the distinct-mass versions of each peptidesequence in a reagent are provided at the same range of concentrations.In some embodiments, each distinct-sequence peptide (e.g. the sum of thedistinct-mass version of the same peptide sequence) is provided at thesame concentration. In some embodiments, each distinct-sequence peptide(e.g. the sum of the distinct-mass version of the same peptide sequence)is provided at a unique concentration. In some embodiments, thedistinct-sequence peptides (e.g. the sum of the distinct-mass version ofthe same peptide sequence) are provided at range of concentrations(e.g., linear or non-linear distribution over a range).

In some embodiments, a reagent comprises buffers, solvents, salts,and/or other additives (e.g., marker (e.g., radiolabel, fluorescent dye,chromophore, mass tag, etc.), etc.) suitable for use in LC and/or MS.Reagents may comprise any suitable buffer (e.g., suitable for use inboth LC and MS), such as Ammonium Bicarbonate, Triethyl ammoniumbicarbonate (TEAB), TAPS, Tris, HEPES, TAE, TES, MOPS, MES, phosphatebuffer, citric acid, CHES, acetic acid, borate, etc. In someembodiments, a reagent comprises additives to promote solubility (e.g.,urea, guanidine HCL, detergents, sugars etc.), stability, etc. of thepeptides within the reagent. In some embodiments, a reagent comprisesone of more solvents, such at acetonitrile, methanol, ethanol, hexane,chloroform, etc.

In some embodiments, the methods and reagents find use in qualitycontrol of any suitable analytic instruments (e.g., LC, HPLC, MS (e.g.,Atmospheric Pressure Ion sources (API), Electrospray or nebulizationassisted Electrospray (ES), Atmospheric Pressure Chemical Ionization(APCI), Matrix Assisted Laser Desorption Ionization (MALDI), etc.),LC-MS, LC-MS/MS etc.). In some embodiments, a reagent is selected,prepared, and/or configured for use with a specific type of instrument(e.g., LC, MS, LC-MS, or LC-MS/MS). In other embodiments, a reagent isspecifically selected, prepared, and/or configured to be used with avariety of instruments. In some embodiments, a reagent is selected,prepared, and/or configured for performing specific quality controlassessments (e.g., calibration, performance evaluation, systemsuitability, etc.).

In some embodiments, the data generated by analysis (e.g., LC, MS, etc.)of a reagent is analyzed manually by a user or a trained specialist. Inother embodiments, software and/or a package of software is provided toperform automated characterization of the reagent analysis. In someembodiments, software analyzes and reports on LC parameters (e.g., peakseparation, peak efficiency, peak height, peak width, peak shape,retention time, etc.) and/or MS parameters (e.g., mass accuracy, massresolution, sensitivity, dynamic range and sampling and/or isolationefficiency). In some embodiments, software correlates peaks in an LCand/or MS analysis to peptides and/or peptide isomers in the reagent. Insome embodiments, software draws conclusions about instrumentperformance based on instrumental analysis. In some embodiments,software compares reagent analyses performed at different time points(e.g., separated by minutes, hours, days, weeks, years, etc.) to monitorchanges in instrument performance. In some embodiments, softwarecalibrates instrument data display/output to offset changes ininstrument performance over time. In some embodiments, the softwareutilizes identifiers for the individual peptides, peptide mix, and/orreagent being analyzed in data analysis. In such embodiments, thesoftware correlates the data with the known physical characteristics ofthe individual peptides, peptide mix, and/or reagent. In certainembodiments, software analysis automatically makes determinationsregarding instrument sensitivity and/or dynamic range. In someembodiments, the software will report on instrument performance history.In other embodiments, the software report on a comparison of performancebetween multiple instruments. In some embodiments, the softwaregenerates a performance score.

In some embodiments, kits are provided that comprise a peptide reagentand one or more of additional reagents, software, container(s),instructions, additional peptide reagent, etc. In some embodiments,suitable aliquots of a peptide reagent are provided in a tube or othersuitable container. In some embodiments, a kit provides multiple qualitycontrol reagents, useful for performing multiple quality controlprocedures.

Experimental EXAMPLE 1 Preparation and Evaluation of Heavy LabeledPeptides

Experiments were conducted during development of embodiments of thepresent invention to develop and evaluate heavy labeled peptides that:are a good indicator of the ability of a column to bind and elute bothhydrophilic and hydrophobic peptides, provide a measure of aninstrument's sensitivity and dynamic range, and provide information onadditional instrument parameters discussed herein.

Selection and design of Peptides: Peptides were derived from acollection of tryptic peptides originating from a human plasma samplethat had been depleted of albumin and IgG. Analysis of the datafacilitated the identification of approximately 8300 unique peptides.Peptides containing W, M, C, P, N, Q, and N-terminal E/D were removedfrom consideration due to stability concerns. Peptides containinghistidine were not considered since they added additional charge to thepeptides. Furthermore, only fully tryptic peptides were considered(those with internal lysines and arginines were removed fromconsideration). Further, only peptides that had the ability toincorporate up to at least 7 stable isotopically-labeled residues andcould allow for a mass difference of at least 4 Daltons per variant wereselected for further consideration.

Peptides were grouped into 6 bins based on retention times. A set ofapproximately 20 peptides per bin were then selected based on signalintensity. These peptides were synthesized in crude form, combined into10 mixtures and analyzed independently on an LTQ-Orbitrap Velos MassSpectrometer. Properties such as retention time, peak shape, signalintensity, most abundant charge state and the ability to produce highquality MS² data were then used in the selection of the last round ofpeptides. The last set of peptides consisted of 28 peptides (spreadacross the 6 bins) which were then used to arrive at the final set ofpeptides. In selecting the final set of peptides (whose concentrationwas predetermined using amino-acid analysis (AAA)), a final set ofpeptides was required to be detectable down to 0.1 fmol (starting from 1pmole; 5 orders of magnitude). In some embodiments, final peptidecandidates were required to be detectable over a very large dynamicrange, sensitive limit of detection and good chromatographic peak shape.In some embodiments, each of the final peptide candidates was requiredto contain at least 7 amino acids that could facilitate the isotopicincorporation of stable, heavy isotopically-labeled amino acids thatwould provide variants with mass differences of at least 4 Daltonsbetween each other such that all 5 peptide isomers within the samplecould be clearly resolved so that an accurate measurement of the peakarea of all isotopes within the peptide envelope could be measured andintegrated.

Experimental Methods: Peptides were synthesized by New England Peptideusing, where appropriate, peptides with stable, heavy isotopes (uniform¹³C/¹⁵N labeling). The following amino acids were required for variouspeptides (see Table 4 below) with the number in parenthesis indicatingthe mass shift in Daltons: (V, +6), (T, +5), (S, +4), (L,+7), (A,+4),(K,+8), (R,+10), (F, +10). Following purification, the peptides weresubjected to amino-acid analysis so the precise molar amount could bedetermined. A mixture of 30 peptides was prepared in 2 formats (SeeTable 5). Both formats contained the same set of 6 peptides with eachpeptide having a set of 5 isomeric variants (30 peptides total). Theisomers were identical in sequence, but contained different heavylabeled amino acids so that they would be distinguished by differingmasses. These masses were used to generate standard curves so as todetermine instrument sensitivity and dynamic range. The specific detailsof the variants and mixtures are given in Table 5 below. Format number 1consisted of peptides in which each set of identical sequence (butvariable with regard to isomer and mass) would differ by a 10-folddifference (either increasing or decreasing amount) of peptide amount.Format 2 was similar to format 1 with the exception that the decreasefrom one mass to the next is a 3-fold decrease. FIG. 16 provides anexample of what the 6 standard curves would look like after correctionfor peak intensities. Additionally, all peptides contained at least onelabeled amino acid to prevent isobaric interference when analyzingsamples from human plasma, etc.

TABLE 4 Preparation of Peptide Stock Solutions Peptide Peptide NumberCode Sequence MW  1 SEQ ID NO. 1 VTSGSTSTSR 1016.532675  2 SEQ ID NO. 1VTSGSTSTSR 1007.515175  3 SEQ ID NO. 1 VTSGSTSTSR 1002.504675  4SEQ ID NO. 1 VTSGSTSTSR  997.494275  5 SEQ ID NO. 1 VTSGSTSTSR 991.480375  6 SEQ ID NO. 2 LASVSVSR  854.532475  7 SEQ ID NO. 2LASVSVSR  846.518275  8 SEQ ID NO. 2 LASVSVSR  839.501075  9SEQ ID NO. 2 LASVSVSR  833.487275 10 SEQ ID NO. 2 LASVSVSR  827.47347511 SEQ ID NO. 3 YVYVADVAAK 1131.644975 12 SEQ ID NO. 3 YVYVADVAAK1123.630775 13 SEQ ID NO. 3 YVYVADVAAK 1117.616975 14 SEQ ID NO. 3YVYVADVAAK 1111.603175 15 SEQ ID NO. 3 YVYVADVAAK 1105.589375 16SEQ ID NO. 4 VVGGLVALR  917.631375 17 SEQ ID NO. 4 VVGGLVALR  910.61427518 SEQ ID NO. 4 VVGGLVALR  904.600375 19 SEQ ID NO. 4 VVGGLVALR 898.586575 20 SEQ ID NO. 4 VVGGLVALR  892.572775 21 SEQ ID NO. 5LLSLGAGEFK 1072.673175 22 SEQ ID NO. 5 LLSLGAGEFK 1062.645975 23SEQ ID NO. 5 LLSLGAGEFK 1055.628775 24 SEQ ID NO. 5 LLSLGAGEFK1048.611575 25 SEQ ID NO. 5 LLSLGAGEFK 1041.594475 26 SEQ ID NO. 6LGFTDLFSK 1068.641075 27 SEQ ID NO. 6 LGFTDLFSK 1058.613775 28SEQ ID NO. 6 LGFTDLFSK 1048.586575 29 SEQ ID NO. 6 LGFTDLFSK 1041.56937530 SEQ ID NO. 6 LGFTDLFSK 1034.552275 Bold residues indicate uniform¹³C/¹⁵N labeling.

TABLE 5 Peptide mixture formats SEQ Fold Decrease ID Peptide [Peptide]from closest NO: Sequence (μM) mass variant Format 2 LASVSVSR 10.00 NA 13 YVYVADVAAK 1.000 10× less 1 3 YVYVADVAAK 0.100 10× less 1 3 YVYVADVAAK0.010 10× less 1 3 YVYVADVAAK 0.001 10× less 1 3 YVYVADVAAK 10.00 NA 1 4VVGGLVALR 1.000 10× less 1 4 VVGGLVALR 0.100 10× less 1 4 VVGGLVALR0.010 10× less 1 4 VVGGLVALR 0.001 10× less 1 4 VVGGLVALR 10.00 NA 1 5LLSLGAGEFK 1.000 10× less 1 5 LLSLGAGEFK 0.100 10× less 1 5 LLSLGAGEFK0.010 10× less 1 5 LLSLGAGEFK 0.001 10× less 1 5 LLSLGAGEFK 10.00 NA 1 6LGFTDLFSK 1.000 10× less 1 6 LGFTDLFSK 0.100 10× less 1 6 LGFTDLFSK0.010 10× less 1 6 LGFTDLFSK 0.001 10× less 1 6 LGFTDLFSK 10.00 NA 1 1VTSGSTSTSR 1.000 10× less 1 1 VTSGSTSTSR 0.100 10× less 1 1 VTSGSTSTSR0.010 10× less 1 1 VTSGSTSTSR 0.001 10× less 1 1 VTSGSTSTSR 10.00 NA 1 2LASVSVSR 1.000 10× less 1 2 LASVSVSR 0.100 10× less 1 2 LASVSVSR 0.01010× less 1 2 LASVSVSR 0.001 10× less 1 2 LASVSVSR 10.0 NA 2 3 YVYVADVAAK3.30 3× less 2 3 YVYVADVAAK 1.10 3× less 2 3 YVYVADVAAK 0.34 3× less 2 3YVYVADVAAK 0.12 3× less 2 3 YVYVADVAAK 10.0 NA 2 4 VVGGLVALR 3.30 3×less 2 4 VVGGLVALR 1.10 3× less 2 4 VVGGLVALR 0.34 3× less 2 4 VVGGLVALR0.12 3× less 2 4 VVGGLVALR 10.0 NA 2 5 LLSLGAGEFK 3.30 3× less 2 5LLSLGAGEFK 1.10 3× less 2 5 LLSLGAGEFK 0.34 3× less 2 5 LLSLGAGEFK 0.123× less 2 5 LLSLGAGEFK 10.0 NA 2 6 LGFTDLFSK 3.30 3× less 2 6 LGFTDLFSK1.10 3× less 2 6 LGFTDLFSK 0.34 3× less 2 6 LGFTDLFSK 0.12 3× less 2 6LGFTDLFSK 10.0 NA 2 2 LASVSVSR 3.30 3× less 2 3 YVYVADVAAK 1.10 3× less2 3 YVYVADVAAK 0.34 3× less 2 3 YVYVADVAAK 0.12 3× less 2 3 YVYVADVAAK10.0 NA 2 3 YVYVADVAAK 3.30 3× less 2 4 VVGGLVALR 1.10 3× less 2 4VVGGLVALR 0.34 3× less 2 4 VVGGLVALR 0.12 3× less 2 Un-bolded residuesindicate uniform ¹³C/¹⁵N labeling

The peptides (30 total) were mixed into a solution and 200 fmol of thissolution was loaded onto a C₁₈ capillary LC Column (75 μm×15 cm). The LCgradient, typically 1 hour, was ramped from 2.5% buffer B (Buffer A—0.1%formic acid in water and Buffer B—acetonitrile/0.1% FA) until 40% B.Mass spectra were collected, in real time, immediately after followingLC. All mass spectra were collected on either a Thermo LTQ-OrbitrapVelos or Q Exactive mass spectrometer operating at MS resolutions of60,000 with calibrated mass accuracies below 3 ppm. Samples wereacquired with a mass range of 350-1200 m/z such that multiply charged(+2/+3) peptides would trigger MS/MS scans.

The peptides of Table 4 were analyzed by LC and MS, both in the formatsdescribed in Table 5 and in other combinations (e.g., isotopologue mixes(e.g., the lightest version of each peptide sequence, second lightest ofeach peptide sequence . . . heaviest version of each peptide sequence)),to assess, for example peptide characteristics and analysis procedures.

The effect of loading time on the binding of the peptides to the LCcolumn was examined (See FIGS. 5A-B). It was determined that shorterloading times (e.g., 2.5 min) were preferential for the binding of allof the peptides, in particular the most hydrophilic sequence (VTSGSTSTSR(SEQ ID NO: 1)).

FIG. 6 depicts an LC analysis of sotopologue mixes (e.g., the lightestversion of each peptide sequence, second lightest of each peptidesequence . . . heaviest version of each peptide sequence) of thepeptides of Table 4. Mix #1 consisted of the “lightest” peptide onlyfrom each set, and each mix was prepared with a progressively heaviervariant. Thus, mix #5 contained the “heaviest” form of each peptide. Thedata indicate that although the peptides contain different types ofstable, heavy labeled amino acids, they are chemically identical (SeeFIG. 6).

A mix of 6 peptide sequences (all equimolar) was subjected tochromatographic gradients (Buffer A=0.1% Formic acid in water and BufferB=0.1% formic acid in acetonitrile; (60 min gradient: 25% B/60 minutes;90 minute gradient: 25% B/90 minutes; 120 minutes: 25% B/120 minutes).Regardless of the gradient, adequate separation and sharp peak shape wasobserved for all six peptide sequences (See FIG. 7).

In order to quantify the amount of a given peptide in a sample mixture(e.g., in order to achieve reliable linear correlations), a correctionfactor was calculated for each of the peptide variants since theisotopic spread (envelope) is different depending on the level ofisotope incorporation (See FIG. 8). To calculate the correctedintensities, the intensities of the visible isotopes are summed, and thesum of the intensities of each is then divided by the intensity of thetallest peak. This value then gives a correction factor for each peptidevariant and thus allows for the normalization of the peak intensity (SeeFIG. 8).

Mass spectra were taken of the various peptide sequences (See, e.g.,FIG. 9). In this example, each different mass version of the peptidesequence was supplied at equal concentration. As a result, approximatelyequal intensities were observed, with mass separations of at least 7Daltons (note that we are observing the doubly charged peptides and thushave m/z spacings of 3.5 Daltons, respectively). Next, all 30 of thepeptides of Table 4 were analyzed by MS in an equimolar mixture. Themolar amounts of each of the peptides (200 fmole) are plotted versus thelog of the corrected signal intensity (FIG. 10). The peptides, whenmixed equally, give equal intensities as predicted.

In addition to the analysis of equimolar samples, format 2 from Table 5,in which different-mass version of each peptide sequence are present atsuccessive 3-fold concentration differences, were analyzed by MS (See,e.g., FIG. 11). Such analysis demonstrates a linear relationship betweenthe five mass variants prepared with a 3-fold drop in log base 3 ofintensity as a function of increasing mass. Similar linear relationshipswere observed for all the sequences tested (See, e.g., FIGS. 12 and 13).Whether the most abundant peptide was the heaviest (FIG. 13) or lightest(FIG. 12) peptide of the given sequence, the same relationship wasobserved. The proportional relationship was observed upon MS analysis ofmixtures of format 1 from Table 5, containing different-mass version ofeach peptide sequence present at successive 10-fold concentrationdifferences (See FIGS. 14-16).

Detection of all peptides in a format 2 mixture was possible when thepeptides were spiked into a complex background of 1 μg/μL yeast trypticdigest (See FIG. 18). An upper bound of quantitation analysis of thepeptide set in yeast background revealed that detection could not extendbelow 200 amol (See FIG. 19), but indicated that a dilution of thespiked sample would facilitate such detection. When the startingconcentration is 200 fmol, the detection limit goes down to 2.4 fmol(See FIG. 19). When the starting concentration is 20 fmol, the detectionlimit goes down to 250 amol (See FIG. 20). When the startingconcentration is 2 fmol, the detection limit goes down to 20 amol (SeeFIG. 21). Thus a method for determining absolute instrument sensitivity(and possibly LOD and LOQ) has been established.

1-25. (canceled)
 26. A peptide mixture comprising four or moredistinct-mass versions of each of five or more distinct-sequencepeptides, wherein three or more of the distinct-mass versions of eachdistinct-sequence peptide comprises one or more amino acids with abovenatural-abundance levels of one or more heavy isotopes, and wherein thedistinct-mass versions of each for each distinct-sequence peptide arepresent at distinct concentrations.
 27. The peptide mixture of claim 26,wherein the peptides are present at concentrations ranging from 10 nM toat to 1 μM.
 28. The peptide mixture of claim 27, wherein the peptidesare present at concentrations ranging from 1 nM (femtomoles permicroliter) to 10 μM.
 29. The peptide mixture of claim 26, wherein thedistinct-sequence peptides are separable by liquid chromatography basedon their different hydrophobicities.
 30. The peptide mixture of claim26, wherein the distinct-sequence peptides are separable by liquidchromatography based on their different charge, size, or hydrophilicity.31. The peptide mixture of claim 26, comprising 5-10 distinct-sequencepeptides.
 32. The peptide mixture of claim 26, wherein the distinct-massversions of any of the distinct-sequence peptides are differentiable bymass spectrometry.
 33. The peptide mixture of claim 26, wherein thedistinct-mass versions of any of the distinct-sequence peptides are theresult of different combinations of stable heavy isotope-labeled aminoacids.
 34. The peptide mixture of claim 33, wherein each of thedistinct-mass versions of any of the distinct-sequence peptidescomprises a different number of uniformly stable isotope-labeled aminoacids.
 35. The peptide mixture of claim 26, comprising 5-10distinct-mass versions of each of the distinct-sequence peptides.